The objective of the proposed research is to elucidate the mechanism whereby ACTH stimulates steroidogenesis in the adrenal cortex, utilizing bovine adrenal cortex cells in monolayer culture as a model system. Cells will be incubated for periods of up to 96 hours in the presence or absence of ACTH. The medium will be changed daily and analyzed for cortisol content by radioimmunoassay. The content of cytochrome P-450 in the cells will be determined spectrophotometrically. Cellular proteins will be radiolabeled with (35S) methionine and the distribution of radioactivity analyzed by PAG-electrophoresis and autoradiography, to determine the effect of ACTH on such radiolabeling. Mitochondrial proteins will be examined in a similar fashion. Total RNA will be extracted from cells and translated in a cell-free translation system. Total translation products will be analyzed in a manner similar to that described for radiolabeled cellular proteins. Newly synthesized cytochrome P-450scc, adrenodoxin reductase and adrenodoxin will be immunoisolated from total translation products to determine whether ACTH treatment results in an increase in the transcription of mRNA specific for these proteins, and to determine whether these proteins are synthesized as precursors. The cellular site of synthesis of these proteins will be determined. The processing of these proteins will be investigated by examining the effect of addition of mitochondria to the cell-free translation system on the conversion of precursor forms to the mature proteins. Possible effects of ACTH on such processing will be investigated. RNA enriched in message specific for cytochrome P-450scc will be prepared and, utilizing this material, a cDNA specific for cytochrome P-450scc will be synthesized. This cDNA probe will be used in hybridization studies to ascertain whether ACTH stimulation of bovine adrenal cortical cells in culture results in an increase in the amount of mRNA specific for cytochrome P-450scc.